Apparatus and method for cell, spore, or virus capture and disruption

ABSTRACT

Embodiments disclose an apparatus and methods for biological sample processing enabling isolation and enrichment of microbial or pathogenic constituents from the sample. A vessel for sample containment and extraction is further disclosed for engagement with a transducer capable of efficient sample disruption and lysis. Together these components provide a convenient and inexpensive solution for rapid sample preparation compatible with downstream analysis techniques.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/074,325, filed Nov. 3, 2014, entitled“APPARATUS AND METHOD FOR CELL, SPORE, OR VIRUS CAPTURE AND DISRUPTION,”and the contents of the foregoing application are incorporated herein byreference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure is directed generally to inventive apparatus andmethods for biological sample processing and analysis. Specifically, thedisclosure relates to a method and apparatus for sample disruption andlysis.

BACKGROUND

Rapid and accurate identification of infectious agents in resourcelimited settings is considered critical for controlling the prevalenceand spread of disease. This is especially the case in high burden areasof the globe where economic and technological limitations constrainefficient disease management. Accurate disease diagnosis and pathogenmonitoring may involve obtaining and isolating biological materials froma subject or patient sample, for example, in the form of sputum, blood,tissue, urine, or other specimen. Sample extraction techniques may thenbe employed to isolate or enrich for nucleic acids, proteins or otherbiological indicators used to detect or identify the presence of apathogen or infectious agent within the sample.

An important area of disease monitoring relates to respiratoryinfections, such as pulmonary tuberculosis. Diagnosis, evaluation, andmonitoring of subjects may involve collection of sputum samples followedby extraction and isolation of pathogen-originating nucleic acidspresent in the sample. For such samples it can be challenging to performsample collection and biomaterial isolation in a manner compatible withdownstream processing and analysis techniques. For example, RNA or DNAassociated with a suspected pathogen may be desirably enriched tofacilitate detection by polymerase chain reaction (PCR), isothermalamplification or other methods. Efficiently breaking down sputum, tissueisolates, and other biological samples can be difficult to perform in asafe and effective manner. Furthermore, low yield isolation protocolsmay increase the difficulty in detecting the presence of suspectedpathogens.

A particular problem exists when attempting to extract and analyzepathogen-identifying nucleic acids from biological samples where theoverall amount of the sample or the amount of nucleic acid present inthe sample is limited. This problem is further exacerbated in resourcelimited settings, such as field applications, rural settings, or inunderdeveloped communities and countries where lab facilities andsophisticated lab equipment may not be readily available. Providingaccess to sensitive and accurate biomolecular testing and analysistechniques in these environments continues to be an important challengeto overcome. In this regard, the present disclosure provides significantadvances in sample processing and analysis techniques that may improvepathogen monitoring and disease management capabilities inresource-constrained regions of the world.

SUMMARY OF THE DISCLOSURE

Embodiments disclose an apparatus for capturing, disinfecting, and/orisolating microbial or pathogenic components of a biological sample. Asample containment vessel or sample collector is provided to isolatedesired components from the sample and may include an integrated filteror separation component. The sample collector may be configured tocouple with a mechanical or sonic transducer capable of transmittingenergy into the sample sufficient to agitate, disrupt, lyse, and/orhomogenize the sample. In various embodiments, the mechanical transducerprovides energy sufficient to lyse the sample constituents releasingbiomolecules such as nucleic acids and/or proteins that may besubsequently detected by downstream analysis techniques.

An innovative coupling mechanism and configuration between the samplecollector and the transducer provides efficient energy transmission intothe sample collector. The transducer configuration permits use ofmechanical, vibrational, or cavitation-inducing energy generated forexample by lower frequency sonic energy as well as higher frequencyultrasonic energy to be applied to the sample providing improved sampleprocessing performance compared to conventional disruption methods. Invarious embodiments, use of lower frequency sonic energy desirablyresults in efficient sample homogenization, lysis and release ofbimolecular constituents while reducing undesirable degradation ofnucleic acids and/or proteins that may be encountered when applyinghigher frequency ultrasound to a sample. An at least partially orsubstantially tapered, infundiblular or conical section of thetransducer provides efficient energy transduction into the samplecollector. The sample collector may be heated and/or cooled while energyis applied to the sample. A lysing fluid may further be utilized todisrupt the sample and release bimolecular constituents of the sample. Afilter integrated into the sample collector may additionally be used tocapture and/or separate desired biomolecular constituents.

The sample containment vessel or sample collector may comprise variousfeatures to facilitate automated or semi-automated sample processing.Additionally, the sample collector and associated instrument componentsmay desirably maintain the sample in an isolated environment avoidingsample contamination and/or user exposure to the sample contents. Anintegrated filter or separation component may facilitate isolation andconcentration of selected sample components, for example, nucleic acidsand/or proteins released from cells or dispersed within the sample.

The methods disclosed herein may be applied in connection withdownstream analysis techniques including polymerase chain reaction orisothermal amplification methods. A removable reservoir integrated intothe sample collector may be used to contain materials such as reactioncomponents and buffers, dilution fluids, sterilization components,preservatives, and/or lysis reagents that can be delivered or mixed withthe sample when the sample has been captured or contained within thevessel. An outlet portion of the collector residing on an opposing sideof the filter may be used to release the isolated sample constituentsfrom the sample collector while other components of the sample areretained in the sample collector. A valve assembly may be used toprovide controllable or selective release of the isolated sampleconstituents for the outlet portion.

In various embodiments, a portion of the sample collector comprises asample capture portion that may be configured in a generally orpartially cylindrical, tapered, or conical shape. The sample captureportion may further be configured with one or more at least partiallytapered, infundiblular or conical sections that couple with or fit intocomplimentary sections of a transducer configured to transmit energyinto the sample containment vessel. In various embodiments, thetransducer transmits mechanical, vibrational, or cavitation-inducingenergy (for example, in the form of sonic or ultrasonic energy) into thesample fluid or media and is used to disrupt or lyse various componentsof the sample. The transducer may further heat or cool the samplecollector and provide selective release of components that have beencaptured by a filter. In various embodiments, the sample is disruptedreleasing at least a portion of the sample's content or constituentsthat may pass through the filter.

In various embodiments, the sample collector may contain constituentscapable of chemically disinfecting the sample or rendering the samplenon-infectious while preserving the integrity of biological constituentssuch as nucleic acids and/or proteins that may be desirably isolated forsubsequent downstream processing and analysis.

In various embodiments, an apparatus is described that permits rapid andsemi-automated decontamination, isolation, and extraction ofbiomolecules, such as nucleic acids and/or proteins from a samplewithout extensive hands-on processing or lab equipment. The samplepreparation apparatus of the present disclosure may further be adaptedfor use with a portable analytical devices and instruments capable ofprocessing and identifying biomolecules, such as nucleic acids andproteins present in the sample.

In various embodiments, the sample collector and various othercomponents of the system can be fabricated from inexpensive anddisposable materials such as molded plastic that are compatible withdownstream sample processing methods and economical to produce. Suchcomponents may be desirably sealed and delivered in a sterile packagefor single use thereby avoiding potential contamination of the samplecontents or exposure of the user while handling. In various embodiments,the reagents of the sample collector provide for disinfection of thesample constituents and permit sample disposal without substantialcontamination risk or remaining infectious or hazardous. The samplecollector may be used in simplified workflows and does not requirespecialized training or procedures for handling and disposal.

In various embodiments, a transducer is configured to couple with thesample collector and facilitates automation of sample processing. Thetransducer is capable of generates mechanical, vibrational orcavitation-inducing energy (for example, arising from sonic orultrasonic vibrations) and efficiently disrupts or lyses components ofthe sample in a safe and isolated manner. The sample collector providesfor subsequent delivery of selected sample constituents and reagents ina manner that reduces chances for cross-contamination and transmissionof potentially infectious materials.

In various embodiments, an apparatus for sample processing is described.The apparatus comprises: a transducer that generates sonic or ultrasonicenergy of a selected frequency, the transducer further comprising aninterface having an at least partially tapered conical surface throughwhich the sonic or ultrasonic energy is propagated; and a samplecollector having a sidewall at least partially complementary to theinterface of the transducer that couples with the interface and receivesat least a portion of the sonic or ultrasonic energy generated by thetransducer and propagates the sonic or ultrasonic energy into aninterior compartment of the sample collector inducing sample cavitationthat disrupts or lyses materials disposed within the sample collector.

The apparatus may further be configured with a transducer that comprisesa recessed portion in which the interface is disposed, the recessedportion of the transducer dimensioned to receive at least a portion ofthe sample collector positioning the interface and sidewall in closeproximity to thereby propagate the energy generated by the transducerinto the interior compartment of the sample collector. Such apparatusmay further be configured with the sample collector dimensioned to beremovably retained in the recessed portion of the transducer with atleast a portion of the sample collector sidewall abuts against theinterface to efficiently propagate the energy generated by thetransducer.

In still other embodiments, a method for biomolecular extraction from asample is described where the method comprises: (a) introducing thesample and one or more fluidic reagents into a sample collector throughan inlet portion of the sample collector; (b) positioning the samplecollector about an at least partially tapered conical interface of atransducer; (c) generating sonic or ultrasonic energy by the transducerthat is propagated into the sample collector through the interfacewherein the energy induces cavitation in the one or more fluidicreagents and results in at least partial disruption or lysis of thesample releasing nucleic acids or proteins; and (d) collecting at leasta portion of the released nucleic acids or proteins through an outletportion of the sample collector.

The apparatus has the further benefit of concentrating biomaterial ofinterest. For example, nucleic acids and/or proteins associated withspores, virus, or bacteria present in the sample may be convenientlyisolated from the original sample material and concentrated. In someembodiments, portions of the sample may be captured on one or morefilters associated with the sample collector 110. Concentration in thismanner may desirably increase the efficiency of the downstream assaysand analysis improving detection sensitivity or providing lower limitsof detection relative to the input sample.

In various embodiments, the automated and semi-automated processingcapabilities of the system simplify sample preparation and processingprotocols. A practical benefit may be realized in an overall reductionin the number of required user operations, interactions, or potentialsample exposures as compared to conventional sample processing systems.This may result in lower user training requirements and feweruser-induced failure points. In still other embodiments, the systemadvantageously provides effective isolation and decontamination of asample improving overall user safety while at the same time preservingsample integrity, for example by reducing undesirable sampledegradation.

Additional objects and advantages of the disclosed embodiments will beset forth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of thedisclosed embodiments. The objects and advantages of the disclosedembodiments will be realized and attained by means of the elements andcombinations particularly pointed out in the appended claims. It is tobe understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the scope of disclosed embodiments, as set forthby the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other embodiments of the disclosure will be discussed withreference to the following exemplary and non-limiting illustrations, inwhich like elements are numbered similarly, and where:

FIG. 1A depicts a cross-sectional view of a sample processing andanalysis apparatus according to various embodiments of the presentdisclosure.

FIG. 1B depicts another cross-sectional view of a sample processing andanalysis apparatus illustrating engagement between a sample collectorand a transducer according to various embodiments of the presentdisclosure.

FIG. 2A depicts a perspective view for an exemplary sample collector andtransducer design in an open configuration according to variousembodiments of the present disclosure.

FIG. 2B depicts another perspective view of the exemplary samplecollector and transducer design in an engaged or closed positionaccording to various embodiments of the present disclosure.

FIG. 3 depicts a cross-sectional view of an exemplary transducer coupledor engaged with a sample collector according to various embodiments ofthe present disclosure.

FIGS. 4A & 4B are representative graphs of average bubble size formed ina liquid over a range of transducer frequencies according to variousembodiments of the present disclosure.

FIG. 4C is a representative graph of relative strength or efficiency ofenergy transfer into a fluidic volume versus transducer oscillationfrequency according to various embodiments of the present disclosure.

FIGS. 5A-C illustrate exemplary embodiments of a sample collector thatmay be adapted for use with a sonic transducer according to variousembodiments of the present disclosure.

DETAILED DESCRIPTION

An exemplary cross-sectional view of a sample processing and analysisapparatus 100 is depicted in FIG. 1A. The apparatus 100 may include asample collector 110 adapted to receive and contain a specimen orsample. In various embodiments, the specimen may comprise a sample orbiomaterial such as bodily fluid, urine, blood, stool, sputum, cells,tissue, spores, or other components obtained from a subject or source tobe processed and analyzed. Various materials, analytes, or isolates maybe desirably recovered from the specimen or sample including by way ofexample, bacteria or other microorganisms, proteins, nucleic acids,carbohydrates, chemicals, biochemicals, particles, or other componentspresent within the sample or specimen.

As will be described in greater detail hereinbelow, the sample orbiomaterial may include infectious, toxic, or otherwise hazardousmaterial that is desirably isolated within sample collector 110 in sucha manner so as to minimize or eliminate exposing the user or handler ofthe sample collector 110 to sample constituents prior to rendering thesample constituents inactive, inert, or in a form that reduces that riskof harm or contamination. As will be described in greater detailhereinbelow, the sample collector 110 design avoids unintended releaseof sample constituents by leakage from the sample collector 110including preventing the escape of aerosols or particulates that mightotherwise present a contamination risk to the user.

The sample or specimen may further comprise solid, semi-solid, viscous,or liquid materials. In certain embodiments, liquid or fluidic reagents(for example, buffers, water, lysis reagents, or other chemicalsolutions) may be added to the sample or specimen to aid in propagationof energy to disrupt or lyse the sample. Similarly, solid materials suchas beads or particulates may be added to the sample or specimen to aidin disruption or lysis. Materials added to the sample may furtherinclude various reagents that facilitate sample dispersion,homogenization, emulsification, or lysis. These materials may furtheract to render the sample inert, inactive, or sterile. In certainembodiments added materials may chemically or physically react withreleased sample or specimen components to preserve or prepare thereleased components for downstream processing.

The apparatus 100 further comprises a cavitation-inducing actuator ortransducer 120 configured to receive and orient the sample collector 110in a desired position within the apparatus 100. The transducer 120comprises a transducer interface 125 whose geometry and size aregenerally configured with at least a portion complementary to the samplecollector 110. In various embodiments, an exterior surface contour orshape of the sample collector 110 is configured to generally align withand/or be positioned against the transducer interface 125 such that thesample collector 110 is seated or located within a portion of thetransducer 120. As will be described in greater detail hereinbelow, theconfiguration and positioning of the sample collector 110 within thetransducer 120 provides a close coupling between the sample collector110 and the transducer interface 125 thereby permitting efficient energytransfer.

The transducer 120 may further be associated with a heater, chiller ortemperature moderating element 130. In various embodiments, a heater isconfigured to adjustably transmit heat to the sample collector 110either directly or indirectly. For example, a heating element 130 maycomprise a controllable resistive heater embedded within or abuttingagainst an armature 135 of the transducer 120 and capable oftransmitting heat energy into the transducer 120. As the transducerarmature 135 is heated this energy may further be transmitted throughthe interface 125 into the sample collector 110. In addition to heatingmeans, the transducer armature 135 may be similarly configured to coolthe sample as desired.

The apparatus 100 may further comprise a temperature sensor 140configured to monitor the temperature of the transducer 120 and/or thesample collector 110. One or more controller boards 145 may receivesignals from the temperature sensor 140 and direct operation of theheater/cooler 130 to achieve or maintain a desired temperature withinthe sample collector 110. In various embodiments, the combined effect ofcontrolled temperature and energy transmission into the sample collector110 enhances the ability of the apparatus to achieve desired agitation,lysis and/or disruption characteristics for processing of sampleconstituents contained within the sample collector.

Energy generation by the transducer 120 (for example, sonic orultrasonic energy) may be provided by one or more coupled piezo devices150 resulting in controllable vibrations or oscillation of thetransducer 120 to provide energy transmission into the sample collector110. Operation of the piezo devices 150 may further be directed by thecontroller(s) 145 which may be configured to direct the frequency ofoperation piezo devices 150 to achieve the desired energy transmissioninto the sample collector 110. In various embodiments, the transducer120 may be secured within the apparatus 110 and provided with a tailmass 155 of appropriate weight or configuration to generate a desired orcharacteristic frequency of oscillation to impart sonic or ultrasonicenergy into the sample collector 110.

In various embodiments, the sample collector 110 comprises an inletportion 160 and an outlet portion 165. The inlet portion 160 may beconfigured to receive a sample or specimen to be processed within thesample collector 110 and secured with a cap or cover 170 which retainsthe sample or specimen within the sample collector 110 while preventingthe escape of solid, liquid, and/or gaseous materials from the samplecollector 110. In various embodiments the cover 170 is secured to thecollector 110 in a screw top, snap top, or other securing/lockingconfiguration with sufficient engagement and retention to prevent theescape of material from the sample collector 110 including avoidingformation of aerosols outside of the sample collector 110 that mayotherwise contaminate the apparatus 100 or release sample constituentspotentially exposing a user to infectious or otherwise dangerousmaterials found in the sample or specimen.

In various embodiments, positive engagement between the sample collector110 and the transducer 120 is maintained by a load member 175. The loadmember 175 may further comprise a spring, button, armature, or othermechanical or electro-mechanical device configured to impart a desiredload or force on the cover 170 and sample collector 110 that urges orprovides a positive engagement or coupling between the sample collector110 and the transducer 120 when the sample collector 110 is suitablypositioned or aligned with the transducer interface 125.

In various embodiments, the sample collector 110 may be configured withthe outlet portion 165 capable of delivering processed sample portionsto other components of the apparatus 100 including for example an assayplate 180. The assay plate 180 may further be configured to receive theprocessed sample and distribute or partition the sample into one or morewells, confinement regions, or chambers associated with the assay plate180. According to certain embodiments, the assay plate 180 may beengaged by a servo or motor 185 capable of moving or rotating the assayplate and facilitating sample distribution within the assay plate 180.

According to various embodiments, a valve assembly 167 may providecontrolled release of processed sample portions to the outlet portion165 of the sample collector 110. In various embodiments, the valveassembly or actuator 167 may be configured in a normally closed positionto retain sample constituents in the sample collector 110 during atleast a portion of the duration energy transfer by the transducer 120.The valve assembly 167 may then be opened according to desiredprocessing protocols to release at least a portion on the processedsample or resulting isolates or constituents. In various embodiments,the valve assembly 167 may automatically open based on achieving adesired or selected pressure within the sample collector 110. Forexample, sample disruption or cavitation may induce a pressuredifferential in the interior of the sample collector 110 causing thevalve assembly 167 to open. Additionally, heat generated in the samplecollector interior may cause the valve assembly 167 to open at aselected temperature or temperature range. In another embodiment, gas orvapor generated in the sample collector may cause the valve assembly 167to open upon achieving a selected pressure or pressure range within thesample collector 110. Gas or vapor generated in the sample collectorinterior may result from reagents added to the sample collector or mixedwith the sample. For example, reagents for generating carbon dioxide,chlorine, chlorine dioxide, nitrogen, or other gases or vapors may beused to actuate the valve assembly 167 and thereby release processedsample constituents in a controlled manner.

In various embodiments, one or more filters 195 may be integrated intothe sample collector 110. As will be described in greater detailhereinbelow, these filters 195 may aid in sample separation and/orisolation to retain selected materials within the sample collector 110while permitting the passage of other materials. For example, sampleconstituents such as cells, tissue, and lysed residual materials may bedesirably retained in the sample collector 110 by the filters 195 whileallowing the selective passage of desired sample isolates such asbacteria, viruses, nucleic acids, carbohydrates, and/or proteins. Thefilters 195 may further have chemical compositions or chemical moietiesdisposed thereon for selectively retaining various sample materials andmay be used to capture and/or separate desired constituents as will beappreciated by those of skill in the art.

The apparatus 100 may further include one or more assay plateheating/cooling elements 190 to maintain the assay plate 180 at desiredtemperature ranges. Configurable heating and/or cooling in this mannermay aid in performing sample reactions using various reagents andprotocols. For example, processed sample received from the samplecollector 110 may comprise concentrated and/or purified nucleic acids tobe subjected to polymerase chain reaction or probe-based nucleic aciddetection techniques within the assay plate 180 for detection and/oridentification of selected sample constituents.

An exemplary sample processing system that may be adapted for use withthe sample collector 110 and sonic transducer 120 for automated orsemi-automated sample processing is described in commonly assigned PCTApplication Serial PCT/US2013/075430 (Publication # WO2014093973)entitled “METHOD FOR CENTRIFUGE MOUNTABLE MANIFOLD FOR PROCESSINGFLUIDIC ASSAYS” to John Nobile, the contents of which are herebyincorporated by reference in its entirety. It will be appreciated bythose of skill in the art that the methods and apparatus of the presentdisclosure may be adapted to other platforms and configurations forsample processing and as such other embodiments and adaptations areconsidered within the scope of the present teachings.

FIG. 1B depicts another cross-sectional view of the apparatus 100depicting an exemplary configuration and engagement between the samplecollector 110 and the transducer 120. Unlike conventional transducerdesigns that may include a relatively small horn or projection with agenerally flat surface for energy transmission into a sample, thetransducer 120 of the present teachings employs an innovative structureincluding an at least partially or substantially tapered, infundiblularor conical recess/cavity 205 associated with the armature 135 that iscapable of receiving or coupling with the sample collector 110. Invarious embodiments, the recess or cavity 205 of the transducer 120 isdimensioned and/or shaped in a manner that permits the sample collector110 to be positioned within or in proximity to the recess 135 whereby asidewall or portion 210 of the sample collector 110 engages with thetransducer 120 at the interface 125. In various embodiments, a closecoupling between the transducer 120 and sample collector 110 is achievedby forming the recess 135 of the transducer 120 to house or contain aportion of the sample collector 110 such that the sample collector 110is at least partially inserted into or resides within the recess 135. Invarious embodiments, the transducer interface 135 may comprise aplurality of surface contours, curvatures, or angles (exemplified byelements 216, 217, 218) that align with or are complimentary to sidewallsurfaces, curvatures, or angles of the sample collector 110 (exemplifiedby elements 226, 227, 228). In various embodiments, configuration of thetransducer 120 with an at least partially or substantially tapered,infundiblular or conical recess 205 desirably improves energytransmission between the transducer 120 and the sample collector 110.

One particular advantage provided by the at least partially orsubstantially tapered, infundiblular or conical transducer design of thepresent teachings is that lower frequency energy or vibrations may beefficiently transmitted into the sample collector 110. Conventionalultrasonic horns are typically configured with a relatively small areafor engagement between the horn and the surface into which energy istransmitted. Such configurations may be necessary in part to insuresufficient propagation of the ultrasonic energy and consequently providelimited or highly focused energy transmission into the sample. Suchmodes of energy transmission may impose significant limitations on howmuch of a sample may receive the energy. In applications where a sampleis to be mixed, disrupted, or lysed, the relatively small or limitedcontact surface between the ultrasonic horn and the sample collectorresults in potentially reducing the overall volume or amount of samplethat can be processed and may further result in incomplete orineffective sample mixing, disruption or lysis.

In accordance with the present disclosure, an innovative transducerdesign overcomes the limitations of conventional transducer designsincreasing the overall surface engagement or contact between thetransducer 120 and the sample collector 110. In various embodiments, theclose coupling of the transducer 120 with the sample collector 110 alongor about one or more surfaces, such as provided by complimentary and atleast partially or substantially tapered, infundiblular or conicaldesigns, desirably increases the overall amount of contact between thetwo components and provides for improved energy transmission into thesample collector 110. Consequently, operations including for example,sample mixing, disruption, or lysis can be performed more efficiently,with less power, and/or more uniformly.

In various embodiments, due at least in part to the improved orefficient energy transfer between the transducer 120 and the samplecollector 110, sample processing operations such as cellular lysis ordisruption to break down or disperse the sample constituents can beachieved without the use of beads or other particulates added to thesample for purposes of enhancing the efficiency of these processes.Avoidance of beads and other particulates also desirably reduces costs,simplifies processing protocols and avoids potential clogging of filtersor membranes that may be used in sample processing.

Despite not requiring particulates for sample processing, the transducer120 and sample collector 110 designs of the present disclosure mayaccommodate use of particulates dispersed within the sample to aid inlysis. In particular, abrasive particles or beads of various dimensionsand compositions may be used according desired processing protocols.Such particulates may comprise polymeric materials such as polystyrene,polypropylene, acrylic-based materials, etc. Additionally, theparticulates may be silica-based, silicone-based, metal, glass, or othermaterials depending on the reactivity of the sample and the desiredapplication. The size or diameter of the particulates may further varydepending upon the application and may, for example, be in the sizerange of approximately 0.1-1 microns in diameter, 1-10 microns indiameter, 10-100 microns in diameter, 100-1000 microns in diameter, orgreater than 1000 microns in diameter. Similarly, the total amount ofparticulates loaded with the sample may vary depending on the amount ortype of sample. In various embodiments, particulates may compriseapproximately 1-10% of the sample volume, 10%-25% of the sample volume,25-50% of the sample volume, or more than 50% of the sample volume.

In various embodiments, the transducer 120 may be configured with morethan one recess or sample collector coupling area 135. For example, thetransducer 120 may be configured to receive two, three, four, or moresample containers 110 into which energy is simultaneously applied.Additionally, the transducer 120 may be configured to couple with otherconfigurations of sample containers capable of discretely retainingmultiple samples such as multiwell plates, microarrays having multiplesample containment regions, or other vessels or consumables for whichsuitable designs for the transducer 120 may be readily configured. Theshape and size of the sample collector 110 and the correspondingcomplementary surfaces of the transducer 120 may be adapted to suit manydifferent applications. For example, the sample container 110 may beconfigured with one or more generally cylindrical, conical, cuboid,pyramidal, or prismatic surfaces. These surfaces may further include anat least partially tapered or narrowed region or section.

In various embodiments, the configuration and dimensions of surfacesfacilitate positioning of the sample collector 110 within the recess orcoupling area 135 of the transducer 120. For example, an at leastpartially tapered or conical sample collector 110 may be approximatelyor roughly positioned above the transducer 120 and deposited within therecess in such a manner that the sample collector 110 and transducer 120readily align or orient with respect to one another to provide improvedcontact or engagement between complimentary elements (depicted forexample by the engagement between elements 216, 217, 218 and 226, 227,228 in FIG. 1B) without having to precisely align the surfaces manually.The guided or self-aligning design of the sample collector 110 withrespect to the transducer 120 is particularly advantageous for use inportable and field instruments where environmental conditions oranticipated usage requirements may not be conducive to precise alignmentby a user.

FIGS. 2A-B depict perspective views for exemplary sample collector 110and transducer 120 designs according to the present teachings. As shownin FIG. 2A, the transducer recess 205 is formed by the interface 125with surface contours (corresponding to elements 216, 217, 218 in FIG.1B) that are complementary to or proportioned in a manner similar to atleast a portion of the sidewalls 210 of the sample collector 110(corresponding to elements 226, 227, 228 in FIG. 1B). As discussedabove, the sample collector 110 may comprise a generally conical taperto facilitate insertion or locating about the transducer recess 205.

The transducer recess 205 may further comprise openings 230, 235permitting convenient insertion or placement of the sample collector 110within or about the recess 205. In various embodiments, at least aportion of the sample collector 110 may extend outside of the transduceropenings 230, 235 in such a manner so that the transducer surfaces donot directly contact the sample collector openings 160, 165. Such aconfiguration desirably isolates or reduces potential exposure of thetransducer surfaces to sample constituents that may be contained in thesample collector 110.

Additionally, the transducer 120 may be configured to transmit energyinto the sample collector 110 at substantially the same time as sampleconstituents or reagents are deposited into or withdrawn from the samplecollector 110. In an exemplary embodiment, sample constituents may beeluted from the sample collector 110 and deposited into a multiwallplate, microarray, or other sample receiving component while thetransducer 120 is in operation. Operation of the transducer 120 andconcomitant energy transmission into the sample collector 110 mayfurther induce, enhance, or encourage sample constituents to exit thesample collector 110. In various embodiments, active energy transferredinto the sample collector 110 by the transducer 120 during elution orremoval of sample constituents may be desirable in instances where atleast a portion of the sample constituents comprise viscous,particulate, or tacky materials.

FIG. 2B depicts an exemplary positioning of the sample collector 110within the recess 205 of the transducer 120 where at least a portion ofthe sidewalls 210 of the sample collector 110 reside in close proximityor are in contact with the transducer interface 125. As previouslydescribed, portions of the sample collector 110 may extend beyond oroutside of the openings 230, 235 of the transducer 120 as shown in theFigure where the outlet portion 165 of the sample collector 110 extendsfrom the recess 205. Configured in this manner the sample collector 135may be coupled directly or indirectly with other apparatus componentsincluding by way of example downstream sample processing consumablessuch as an array, microwell, sample receiving module, or other devices(not shown in the Figure) as will be understood by those of skill in theart. In various embodiments, engagement of the sample collector 110 withthe transducer 120 may include a latch, spring, or screw member thatsecures or positions these components in a desired orientation or with adesired positive pressure or coupling force to insure efficient energydistribution into the sample collector 110. The securing or positioningcomponent may be integrated into the sample collector 110 and/or thetransducer 120 or alternatively be provided by an exterior member suchas the previously described spring or latch 175 shown in FIG. 1A.Providing a securing component in this manner may be advantageous wherethe apparatus is a portable or field operative device that is subject tojarring, external vibrations or other conditions that may disengage ordislocate the sample collector 110 from the transducer 120 in undesiredmanners and/or before sample processing is complete.

FIG. 3 depicts another cross-sectional view of the transducer 120 withthe sample collector 110 in a coupled or engaged position. Asillustrated, one or more transducer interface surfaces, for exampleshaped or configured as one or more at least partially tapered,infundiblular or conical sections, are in close proximity or engagedwith the sidewalls 210 of the sample collector 110 providing efficientenergy transfer into the sample collector interior and sampleconstituents and/or reagents contained therein. Various modes ofvibration or energy transfer may be induced within the transducer 120 asillustrated by the motion or force vectors 305. In addition to generallylinear motion or vibration, circular or other patterns or combinedpatterns of motion may be imparted to the sample collector 110 by thetransducer 120.

Ultrasonic energy waves have a frequency above 20 kHz. Ultrasonicdisruptors employing a probe or horn-based probe for energy transmissionmay operate in a frequency from 50 kHz to 500 kHz, and frequencies mayextend into the range from about 0.5 MHz to about 5 MHz. In variousembodiments, the transducer designs and methods of the presentdisclosure are configured to operate both at sub-ultrasonic frequenciesand also ultrasonic frequencies. In various embodiments, sonicfrequencies may be sub-20 kHz, sub-15 kHz, sub-10 kHz, sub-5 kHz, orless. According to the present disclosure, the various transducerconfigurations described herein desirably provide efficient sonic energytransmission and propagation properties. Conventional probe orhorn-based designs do not propagate lower frequency sonic energyefficiently and as a result sonic energy has not been widely usedconventionally. This may be due in part to the lower efficiencydisruption and lysis properties of sonic energy when applied byconventional transducer designs. The transducer designs of the presentdisclosure overcome the limitations of conventional transducer designsand provide for efficient sonic and ultrasonic energy transmission andpropagation properties.

Another significant advantage realized using relatively low frequencysonic energy is that the size of cavitation bubbles formed in a liquidmedium may be increased relative to ultrasonic frequencies. Cavitationbubbles arise where vibrational or mechanical energy propagates througha liquid or fluidic medium. Cavitation bubbles are formed and grow whena liquid is put in significant state of tension. Acoustic pressure wavestransmitted by energy transmission from a transducer result in theliquid undergoing a compression and rarefaction cycle. Duringrarefaction, pressure in the liquid becomes negative and when thenegative pressure falls below the vapor pressure of the fluid medium,the energy wave may cause voids or cavitation bubbles to form in themedium. The size of the bubbles formed affects the efficiency of sampledisruption or sample lysis.

In various embodiments, it is desirable to generate relatively largecavitation bubbles to enhance agitation, disruption, and/or lysisproperties of energy transmitted by the transducer. According to thepresent disclosure, sonic energy may be efficiently transmitted into anat least partially liquid, viscous, or fluidic sample to create largercavitation bubbles than conventional ultrasonic disruptors. It will beunderstood, that while the transducer designs of the present disclosureare particularly suitable for sonic energy generation and transmissioninto a sample, these designs may be adapted for use with higherfrequency acoustic and mechanical energy ranges such as ultrasonic ormegasonic applications without departing from the scope of the presentteachings.

In various embodiments, suitable frequency parameters for the transducermay be in the range of approximately 20 kHz or less. In someembodiments, the transducer may be operated in the range ofapproximately 17.5 kHz. In still other embodiments, the transducer maybe operated in the range of 15 kHz or less. For the operatingfrequencies described above, the transducer may be operated atapproximately 400 volts or less, 200 volts or less, or 100 volts orless. Efficient sample disruption may further be provided by operationof the transducer over brief time intervals. For example, energy may beapplied to a sample in multiple short intervals for example, between1-10 intervals of approximately 60 seconds or less. In otherembodiments, between approximately 4-8 intervals of transducer operationfor 20-40 seconds may be used. In some embodiments, relatively shortpauses between application of energy to the sample may take place. Forexample, pauses between approximately 1-5 minutes or less between energyapplication intervals may be applied preventing sample over-heating. Insome embodiments, pauses of approximately 1 minute or less with energyapplication intervals of approximately 1 minute or less in a frequencyrange of approximately 20 kHz or less may result in sufficient lysis ofa sample. For example, 4-6 intervals of transducer operation for 30seconds to 1 minute at approximately 15-20 kHz cycles with 30 sec-1minute pause cycles may result in lysis efficiency of over 90% forbacterial samples such as Mycobacterium smegmatis and Tuberculosismycobacterium.

In various embodiments, a sample to be processed may be inserted into anupper portion or sample retention region 310 of the collector 110. Thesample along with various reagents, disinfectants, buffers, and otherconstituents may then be subjected to a desired disruption or lysisprotocol. Operation of the transducer 120 may occur at various desiredfrequencies, temperatures, and/or time intervals such that the sample ismixed with other constituents over a first interval and lysis or sampledisruption takes place over a second interval. As previously described,sample disruption or lysis may release biomolecules or otherconstituents into a fluidic volume of the sample retention regions 310.As will be appreciated by those of skill in the art, numerous protocolsand/or methods may be adapted for use with the collector 110 includingmultistep protocols such as those which involve a sample lysis stepfollowed by a capture and/or elution step to isolate compounds,chemicals, or sample constituents of interest.

A filter, size exclusion membrane, selective chemical/molecular capturesurface, or other separation component 320 may be integrated into thesample collector 110. In various embodiments, the separation component320 may be positioned generally below the sample retention region 310and provide the ability to separate, capture, or selectively elutedesired chemicals, biomolecules, or other constituents from the sample.Certain sample constituents may further be retained in the collector 110and others passed through the collector outlet 165 thereby isolating,purifying and/or concentrating desired materials from the sample. Aspreviously discussed, and in various embodiments, nucleic acid and/orprotein constituents may be isolated from a biological specimen andeluted from the collector 110 into a separate array, microwell, or othersample collection device for further processing and analysis.

The transducer configurations of the present teachings desirably provideimproved energy transmission properties compared to conventionalultrasonic disruptors. In certain configurations, the structure andshape of the transducer interface 125 provide enhanced energytransmission properties for both lower frequency sonic energy vibrationor mechanical oscillation and ultrasonic energy. An innovative aspect ofthe transducer design permits relatively low frequency sonic energy(e.g. sub-ultrasonic range) to be transmitted into the collector 110 inan efficient manner facilitating sample disruption and lysis.

FIGS. 4A-C illustrate exemplary characteristics of sonic energy transferthat provide for improved sample processing according to the presentteachings. Vibrational or mechanical transmission into a liquid samplemay result in the formation of bubbles within the liquid. The size ofthe bubbles may further affect the mixing, shearing, or lysis propertieswithin the sample when subjected to a selected range of energytransmissions. In various applications, it is desirable to generaterelatively large bubbles to improve the energy transmission propertiesinto the sample and/or to generate a desired mechanical effect (e.g.disruption, lysis, or sheering) on the sample constituents. For example,when disrupting or lysing thick or viscous samples such as sputum,blood, or tissue it may be desirable to maintain a bubble populationwith a size larger than typically provided by ultrasonic energytransmission.

According to the present teachings, larger bubbles and/or more efficientmixing and/or lysis of a sample can be achieved using lower frequencysonic energy transmission as compared to ultrasonic energy transmission.Sonic energy transmission may also be less disruptive or damaging tovarious sample constituents such as nucleic acids and/or proteinsreducing the overall fragmentation of these molecules and preservingtheir integrity during sample processing.

FIGS. 4A & 4B are representative graphs of average bubble size formed ina liquid over a range of transducer frequencies. At higher frequencies(approximately in the range of 100 kHz or more) bubble size is markedlyreduced compared to bubbles formed at lower frequencies (approximatelyin the range of less than 100 kHz). In particular, bubbles formed in therange of 20 kHz or less may be several times the size or volume ofbubbles formed at higher frequency ranges. Larger bubbles may improvethe sample processing characteristics and energy transmission propertiesof the transducer obviating the need to include beads or otherparticulates in the sample or carrier fluid which are conventionallyused to enhance mixing and/or lysis.

FIG. 4C is a representative graph of relative strength or efficiency ofenergy transfer into a fluidic volume versus transducer oscillationfrequency. At higher frequencies (e.g. in the ultrasonic range) overallenergy transmission is relatively low as compared to lower frequencies(e.g. in the sonic range). Notably, at frequencies lower than 20 kHz,overall energy strength can be several orders of magnitude greater thanat frequencies higher than 100 kHz. An innovative aspect of the presentteachings is the recognition that lower frequency energy transmissionswhen suitably applied to the sample may therefore give rise to improvedsample disruption and lysis properties. Thus, by configuring thetransducer 120 to accommodate efficient low frequency energytransmission into the sample collector 110, enhanced sample disruptionand lysis may be achievable relative to conventional ultrasonic energyapplications.

FIGS. 5A-C illustrate exemplary embodiments of a sample collector 110that may be adapted for use with the transducer designs of the presentdisclosure. A cap or cover 170 and a waste or liquid reservoir 505 maycouple or engage with the sample collector 110. In various embodiments,these components form an integrated device that may be used to collect,house, and/or store a sample to be later processed using the transducer120. FIG. 5A depicts the sample collector 110 in a closed or sealedarrangement with the cover 170 and liquid reservoir 505. In thisconfiguration, sample material may be desirably contained within asample retention volume of the sample collector 110 and isolatedpreventing contamination or leakage of the sample constituents.

As shown in the open sample collector arrangement in FIG. 5B, the cover170 may further comprise a plunger or button 510 used to mix or positionsample constituents and/or reagents within the internal collectorvolume. In certain embodiments, the cap or cover 170 or a portion of thecollector 110 may be adapted to house one or more reagents that aredesirably mixed with the sample constituents. For example, the cover 170may contain, store, or sequester one or more different reagents that arecontained in one or more compartments 515. Alternatively, thesecompartments 515 may be integrated into the collector 110. The reagentsmay further be isolated, preserved, and/or separately contained using anadhesive or heat sealed film or foil or by other means as will beappreciated by those of skill in the art.

In various embodiments, the plunger 510 and/or other components of thesample collector 110 may include a piercing or opening member 520capable of releasing the reagents when the sample is added to thecollector 110. The waste or liquid reservoir 505 may further providesufficient support to act as a stand or holder when fitted to the samplecollector 110 and in various embodiments encloses the collector opening165, filters 195, and other components of the sample collector 110. Thereservoir 505 may also be snapped or screwed onto the sample collector110, for example with with a non-airtight fit to the bottom opening 165of the sample collector allowing venting.

One or more of the filters 195 may be positioned in assemblies about thecollector 110 and along with the cover 170 and/or liquid reservoir forma substantially air or liquid impermeable seal or closure preventingundesired leakage from the collector 110. In various embodiments,selected components or parts such as the cover 170 or reservoir 505 mayengage with the collector 110 using mated threads and may be sealed forexample by precision mating surfaces or elastomeric seals. In variousembodiments, the filters 195 may be designed to catch or retainbiomaterials such as spores or bacteria of a certain minimum size, forexample, above 0.2 um, but let liquid, particulates, and other reagentspass through when sufficient hydrostatic pressure is applied. Suchhydrostatic pressure may be generated by the plunger 510 or by variousother means including cavitation or heating of the sample by thetransducer 120.

In various embodiments, the sample collection device 110, including theone or more filter assemblies 195, may be designed to directly acceptthe sputum or other biological sample from a subject. As shown in FIG.5C, in various embodiments, after the sample is captured in the samplecollector 110, a first reagent supply cap 525 may be coupled to thesample collector 110. Such an approach desirably allows reagents to beadded to the sample or specimen in a safe and non-disruptive mannerreducing the likelihood of user or device contamination and furtheravoids aerosol formation. As the reagent cap 525 is secured orpositioned on or within the collector 110 one or more reagent reservoirswithin the cap 525 may be punctured or opened by piercing features 520in the sample collector 110 as previously described.

In various embodiments, the reagents may be designed to mix or reactwith the sample for purposes of preservation or performing chemicalreactions. For example, a sputum sample may be mixed with reagentsselected to dilute and/or liquefy the sputum, neutralize or lysemicrobes present in the sample, and/or render the sample non-infectious.Another function of the one or more reagents may, in variousembodiments, engage in a chemical reaction that will evolve a gas togenerate an increased pressure within the sample collector 110. Theincreased pressure may be sufficient to push or drive portions orsubstantially all of the sample through the one or more filters 195 intothe liquid reservoir or tank 505. In various embodiments, passage of thesample through the one or more filters 195 may trap or isolate some orsubstantially all of the desired microbes or other materials on thefilters 195. An alternative method of passing the liquefied samplethrough the filters 195 is for the reagent delivery cap 525 to contain aplunger 510 that would be actuated by the user at such time when thereaction is complete.

While the principles of the disclosure have been illustrated in relationto the exemplary embodiments shown herein, the principles of thedisclosure are not limited thereto and include any modification,variation or permutation thereof.

1-16. (canceled)
 17. A method for biomolecular extraction from a sample,the method comprising: a) introducing the sample and one or more fluidicreagents into a sample collector through an inlet portion of the samplecollector; b) positioning the sample collector about an at leastpartially tapered conical interface of a transducer; c) generating sonicor ultrasonic energy by the transducer that is propagated into thesample collector through the interface wherein the energy inducescavitation in the one or more fluidic reagents and results in at leastpartial disruption or lysis of the sample releasing nucleic acids orproteins; and d) collecting at least a portion of the released nucleicacids or proteins through an outlet portion of the sample collector. 18.The method of claim 17, wherein a portion of the energy induces thereleased nucleic acids or proteins to pass through the outlet portion ofthe sample collector.
 19. The method of claim 18, wherein the samplecollector further comprises a cap that seals the inlet portion of thesample collector and creates positive pressure within the samplecollector when the energy is applied forcing the released nucleic acidsor proteins to pass through the outlet portion.
 20. The method of claim17, wherein the energy generated by the transducer is less than 50 kHz.21. The method of claim 20, wherein the energy generated by thetransducer is less than 20 kHz.
 22. The method of claim 17, wherein theis generated by one or more piezo devices coupled to the transducer. 23.The method of claim 17, wherein the sample comprises cells, spores, orviruses.
 24. The method of claim 17, wherein the transducer generatesheat and cavitation-inducing energy propagated into the samplecollector.
 25. The method of claim 17, wherein the transducer comprisesa recessed portion in which the interface is disposed, the recessedportion of the transducer receiving at least a portion of the samplecollector positioning the interface and sidewall in close proximity tothereby propagate the energy generated by the transducer into the samplecollector.